This invention relates to the preparation and stabilisation of cell suspensions, and more particularly to a novel method for preparing and stabilising cell suspensions, and to the use of novel stabilised cell suspension preparations in the quality control of analytical techniques such as UV microscopy and flow cytometric leucocyte immunophenotyping techniques, immobilised antigen/antibody detection systems and haematology analysers, and blood monitoring techniques such as zinc protoporphyrin (ZPP), red cell folate and blood glucose measurements.
UV microscopy and flow cytometry are techniques used in the diagnosis of haematological malignancies. They are also used to monitor the progress of patients infected with the Human Immunodeficiency Virus (HIV) whether asymptomatic or suffering from ARC or full-blown AIDS. Quality control (QC) of these two techniques is extremely important to arrive at the correct diagnosis and to monitor effective therapeutic regimes. The current QC methods use freshly drawn blood or microspheres coated with a fluorochrome.
The use of fresh blood on a daily basis fails to provide the information on day to day variation of the technique or equipment. Furthermore, fluorochrome coated microspheres, though providing a day to day monitor of the flow cytometer's performance cannot be used for UV microscopy work. In addition, they cannot be used to provide quality control for the labelling techniques of leucocytes.
Fixation of normal leucocytes, utilising compounds such as paraformaldehyde, although giving stability for 5-7 days increases cellular autofluorescence. This makes the preparation unsuitable for use as a long term quality control material. Furthermore, the lysis of red calls by the whole blood lysing technique requires a preparation that will quality control this procedure. The current methods of fixing the leucocytes inhibit this lysing procedure resulting in a significant increase in debris that interferes with the tests.
Other QC equipment requiring the use of whole blood samples or blood products for calibration include haematology analysers where currently fixed blood from donkeys and turkeys is used because a suitable source of stabilised human blood is not available. Zinc protoporphyrin (ZPP) and red cell folate monitoring techniques also require a fresh suspension of red blood cells for calibration, again because a suitable stabilised source is not available. Finally the lack of a stabilised source of whole human blood for calibration purposes limits the possibility for diabetics to carry out blood sugar monitoring at home.
In International Application No. WO 91/17436, the entire disclosure of which is incorporated herein by reference, there is described a blood diluent and lysing agent for differential determination of white blood cells (leucocytes) in which the stabilising agent is diazolidinyl urea. Such leucocyte preparations have not been suggested as flow cytometric preparations possibly because they have insufficient stability and lack certain specific antigenic activity for those routine quality control procedures which need to assess-results from a large number of laboratories.
International Application No. WO92/19951 discloses the use of diazolidinyl urea, imidazolidinyl urea, dimethylol-5,5-dimethylhydantoin, dimethylol urea, 2-bromo-2-nitropropane-1,3-diol and quaternary adamantane as tissue fixatives which are free of aldehydes. The formulations may contain inter alia mordants such as zinc, calcium, barium and chromium salts. It is not suggested, however, that any of these salts have stabilising properties.
G81563839 provides a process for preparing a stabilised preparation of erythrocytes, wherein a suspension of erythrocytes in an aqueous isotonic solution is reacted with, simultaneously or successively in any sequence, an aliphatic aldehyde and a water-soluble salt of 2- or 3- valent chromium. In the examples, the erythrocytes are treated with formaldehyde and glutaraldehyde, followed by an aqueous solution of chromium III chloride.
It has been found that cell suspensions containing erythrocytes treated in this way are no longer suitable for use in a whole blood lysing technique, and thus the process of GB1563839 cannot be used to prepare stabilised whole blood samples, for example, for flow cytometry quality control.
GB2001757 describes a method of preparing a stable suspension of blood platelets which comprises adding chromium ions to a platelet-rich plasma and adding at least one organic aldehyde to the chromium-platelet-rich plasma mixture. In the examples, the platelet-rich plasma is treated with solutions of potassium dichromate and glutaraldehyde. This process, if used on leucocyte cells, substantially increases autofluorescence and renders the suspension unsuitable for use in flow cytometry.
The entire disclosures of the above-mentioned patent and applications are incorporated herein by reference.
It will be appreciated from the above, that there is a need for an improved method for stabilising cell suspensions, particularly of whole blood and blood products, giving a cell preparation that is still capable of lysis, for a variety of quality control, monitoring and calibration applications.
In UK Patent Application No. GB2279653 it is disclosed that certain heavy metal compounds can stabilise leucocyte preparations formed from lysed whole blood, and that such stabilised preparations can be added to leucocyte-deplated blood to provide a stabilized whole blood preparation with high antigenic activity. The entire disclosures of these applications are incorporated herein by reference.